Molecular cloning and expression of Campylobacter pylori species-specific antigens in Escherichia coli K-12.

A gene bank of Campylobacter pylori DNA in Escherichia coli was constructed by cloning Sau3A-cleaved DNA fragments into the bacteriophage vector lambda EMBL3. The expression of C. pylori antigens was determined by screening the gene library with adsorbed C. pylori whole-cell rabbit antisera. One recombinant clone which reacted positively (lambda CP2) was studied further. Immunoblot analysis with lambda CP2 showed a polypeptide band of 66 kilodaltons (kDa) reacting antigenically with the adsorbed antiserum. Extraction of DNA from lambda CP2 and digestion with SalI revealed a DNA insert of 17 kilobases (kb). Subcloning with SalI and the E. coli vector pUC18 showed that the DNA also encoded a 31-kDa antigen. The cloned antigens were shown by immunoblotting to have the same molecular weight in E. coli as in C. pylori and to be present in all C. pylori strains. Antiserum was raised against the cloned polypeptides and found to react only with C. pylori when analyzed by dot blotting and indirect immunofluorescence. The cloned antigens were determined to be expressed from the pUC18 lac promoter. The DNA encoding these antigens was radiolabeled with 32P and found to hybridize only to C. pylori strains. Immunoblotting with affinity-purified polyclonal antibody to the urease enzyme of C. pylori revealed that the cloned antigens may be part of the urease enzyme.     

Amplification of unknown DNA sequences by sequence-independent nested polymerase chain reaction using a standardized adaptor without specific primers.

A new procedure for sequence-independent PCR amplification of DNA fragments is described. DNA from pUC18 plasmid was used as a test DNA. It was digested with a frequently cutting restriction enzyme (Sau3A), generating sticky ends. The DNA was ligated to a synthetic, non-phosphorylated adaptor and subsequently amplified in a nested PCR using two oligonucleotides with sequences derived from the adaptor. As little as 1 fg of pUC18 DNA could be detected by this procedure. The product was analyzed on a gel and hybridized with a pUC18-specific probe. The sequence-independent nested PCR was repeated with different amounts of pUC18 DNA in the presence of an excess of non-specific DNA. In these experiments, pUC18 DNA fragments were amplified in a concentration-dependent manner. After hybridization with a digoxigenin dUTP-labelled pUC18 DNA probe, 1 fg of pUC18 DNA could still be detected. This method allows rapid screening of blood for low titred and mutated viruses in which primer binding sites are not conserved.     

Influences of polychlorinated dibenzofuran on pUC18 plasmid DNA

We investigated an effect of polychlorinated dibenzofuran (PCDF) on pUC18 plasmid DNA. The mixtures of pUC18 and PCDF were introduced into the competent cells derived from E. coli. The competent cells were cultured on Luria-Bertani agar plate containing ampicillin. The number of colonies was not decreased in compared with it of E. coli inserted non-treated pUC18. The pUC18 re-extracted from the E. coli with PCDF-treated pUC18 were digested with 15 kinds of restriction enzymes and agarose gell electrophoresed. As the results no changes were found in compared with non-treated pUC18. Those findings suggested that the PCDF did not react with pUC18.     

旋毛虫抗原分子克隆及其T细胞和B细胞表位预测

目的 克隆旋毛虫肌幼虫Mr21 000抗原蛋白基因(Ts21),预测其T细胞和B细胞表位.方法 旋毛虫(河南地理株)感染小鼠后收集肌幼虫,提取肌幼虫总RNA,应用RT-PCR技术获取目的基因Ts21,构建重组质粒pUC18-Ts21并测序,应用生物信息分析软件预测其T细胞和B细胞表位.结果 成功构建克隆载体pUC18-Ts21.旋毛虫Mr21 000抗原的T细胞表位位于第9～23、135～150位氨基酸位点;B细胞表位位于第31～35、53～56、98～104、124～130、133～157、160～172位氨基酸位点.结论 成功克隆了旋毛虫河南地理株肌幼虫Mr21 000抗原的编码基因,T细胞和B细胞表位预测结果表明该抗原蛋白具有较好的抗原性,可作为旋毛虫病免疫诊断和预防的候选抗原.     

Repetitive DNA probes for the detection of Babesia equi

This report describes DNA probes for the identification of Babesia equi. A genomic library of B. equi was constructed in pUC13. Several clones were identified that hybridized strongly to B. equi DNA. Clone pBE33 hybridized specifically to B. equi DNA and did not hybridize to horse DNA nor to DNA from Babesia caballi, Babesia bovis or Babesia bigemina. Two subclones of pBE33 (pSB20 and pEH21) containing B. equi repetitive sequences, could detect 0.49 ng and 0.97 ng B. equi DNA, respectively.     

Specific interaction of the SV40 T antigen-cellular p53 protein complex with SV40 DNA

Three forms of the simian virus 40 (SV40) large tumor antigen (T antigen), free T antigen from infected monkey cells, T antigen-p53 complex from virus-infected mouse cells, and T antigen-p53 complex from SV40-transformed mouse cells, were examined for their abilities to bind specifically to SV40 DNA. Employing an immunoassay that detected T antigen and/or p53 protein bound to restriction fragments of viral DNA, it was shown that all three of these forms of SV40 T antigen bind preferentially to the restriction fragment of SV40 DNA containing the nucleotide sequence at the origin of viral DNA replication. Free p53 protein alone failed to bind to SV40 DNA in this assay. These results indicate that the SV40 T antigen-p53 protein complex displays the same SV40 DNA binding specificity as free T antigen obtained from a productive infection.     

EXPRESSION OF HUMAN CD59 ANTIGEN ON MOUSE NIH3T3 AND EL-4 CELLS CONFERS PROTECTION AGAINST HUMAN COMPLEMENT ATTACK

CD59分子是广泛分布于各组织细胞表面的18kDa糖蛋白,通过肌醇磷脂酰聚糖(GPI)锚固于细胞膜,具有抑制同源补体攻膜复合物(MAC)形成和参与介导T细胞活化等多种功能.本文应用RT-PCR方法,从Jurkat细胞的总RNA中扩增得到396bp的cDNA片段,经测序证实该片段包括25aa信号肽在内的全部的CD59编码序列.进一步将此CD59的cDNA重组于逆转录病毒载体pLXSN,电穿孔转染PA317细胞,并用病毒上清感染小鼠成纤维母细胞NIH3T3及小鼠胸腺瘤细胞EL-4.经G418加压筛选,FACS检测获得表达CD59的阳性细胞克隆.补体杀伤实验结果表明:表达GPI-型CD59分子的NIH3T3和EL-4细胞对人血清补体溶破的抵抗作用较空载体转染的非表达细胞明显增强.证实了用逆转录病毒载体可成功地将人CD59基因导入异源细胞,使表达CD59的异源细胞获得抑制人补体溶破的功能.本研究为探讨CD59分子与细胞活化的关系及其信号转导机制建立了良好的细胞模型,并为进一步应用于异种器官移植,或对由于CD59遗传缺损所致PNH进行基因治疗奠定了基础.     

人CD34抗原基因克隆及鉴定

ＣＤ３４抗原是一个阶段特异性白细胞分化抗原，在早期造血调控方面起着重要的作用，本文采用ＲＴ－ＰＣＲ方法，从高效表达ＣＤ３４抗原的ＫＧ－ｌａ细胞系中成功地克隆出ＣＤ３４抗原全长ｃＤＮＡ，并对重组基因进行了酶切鉴定和序列分析，结果表明除胞内区有一碱基改变，其它序列与报道完全一致。     

Recombinant DNA related to hepatitis B and human immunodeficiency viruses in mononuclear cells of patients with AIDS

Sixty-eight of 73 patients with human immunodeficiency virus (HIV) infection were positive when tested for the presence of hepatitis B virus (HBV)-related DNA sequences in peripheral blood mononuclear cells (PBMCs) by the dot blot method. Twenty-two of the positive DNAs were examined by Southern hybridization and all exhibited a 3.2 kb extrachromosomal DNA fragment that hybridized to HBV DNA. This DNA was isolated from agarose gels and cloned into the EcoRI site of pBR322 DNA. The cloned DNA (pHBI) hybridized to both HBV DNA and HIV cDNA; HBV DNA did not hybridize to HIV cDNA under the same conditions. The results of restriction enzyme analyses indicated that pHBI contains: 1) a large deletion of HBV sequences spanning the 3' end of the HBV surface antigen gene; 2) a small deletion near the 5' end of the HBV core antigen gene; and 3) a region of homology to a one kb central section of the HIV pol gene. These data suggest that the 3.2 kb DNA found in the PBMCs is a natural recombinant between HBV and HIV DNAs raising the possibility not only that this DNA plays a role in the pathogenesis of AIDS but also that other viral recombinant DNAs may be pathogenic in human disease.     

Circular plasmid DNAs from the red alga Gracilaria chilensis

Summary Total cellular DNA extracted from eight red algal species (from the genera Gracilaria, Gracilariopsis, Porphyra and Gymnogongrus) was centrifuged on Hoechst dye/CsCl gradients. In five species, plasmid-like DNAs banded with the A+T rich organellar DNAs in the CsCl gradients. Based on their electrophoretic migration in different agarose gels, the plasmid-like DNAs are circular. This is the first report of putative plasmid DNAs in the red algae outside the genus Gracilaria. Two similar Gracilaria chilensis plasmid-like DNAs of 3.8 and 3.4 kb (GC2 and GC3) were cloned in pUC19. The cloned GC2 DNA did not hybridize to either the organellar or nuclear genomes of G. chilensis, suggesting that GC2 is a true plasmid. GC2 did hybridize to the plasmid of one other red algal species, Gracilaria sordida.     

一株多重耐药肺炎克雷伯菌KF3的质粒研究

目的 通过对肺炎克雷伯菌KF3质粒DNA全序列测定,从基因组水平研究质粒DNA的结构、功能基因和与宿主菌耐药相关性.方法 碱裂解法提取质粒DNA,构建质粒DNA文库并测序.采用Phred/Phrap/Consed软件包进行序列拼接,Glimmer软件预测开放阅读框架(ORF)及功能分析.结果 构建包含3个质粒DNA的pUC18文库和Fosmid文库,测序获得3个质粒全序列.功能注释分析发现3个质粒均为可接合转移质粒,编码大量耐药相关基因.结论 肺炎克雷伯菌KF3的3个质粒都是可接合转移质粒,将耐药基因在细菌间进行水平转移,造成了耐药菌的播散.     

Presence of Epstein-Barr virus DNA in tonsillar tissues

Sera from 48 tonsillar carcinoma (TC) patients, 48 matched controls and 16 recurrent exudative tonsillitis (RET) patients were examined for the presence of Epstein-Barr virus (EBV) associated nuclear antigen (EBNA), early antigen (EA) and virus capsid antigen (VCA). Higher prevalence and significantly higher antibody titres against all three EBV-associated antigens were observed in TC patients in comparison with controls and RET patients. Patients suffering from anaplastic TC had higher titres of antibodies against VCA and EA than TC patients with other histological diagnoses. Five out of 11 TC biopsies obtained from 9 patients were positive for EBV DNA at levels of 0.17, 4 to 5, 15 to 18 and in two cases 3 EBV genome equivalents per cellular genome. Among 16 RET patients, 4 were found positive at levels not exceeding 2.17 EBV genome equivalents per cellular genome. Higher titres of antibody against all EBV antigens were found in TC and RET patients with EBV DNA-positive tonsillar tissue than in those with EBV DNA-negative tonsillar tissue.     

Hybridization of herpes simplex virus DNA and human ribosomal DNA and RNA

A small DNA segment from the inverted repeats at the termini of the unique long sequence region of herpes simplex virus DNA was found to hybridize with human 28 S ribosomal DNA and RNA but not 18 S ribosomal DNA and RNA. The hybridization occurred under stringent conditions and was not blocked by nucleic acids high in guanine plus cytosine content. These data strongly suggest that the hybridization represented authentic base sequence homology.     

Antigen Restricted Hybridization Between Antigen Primed Macrophage and Thymic RNA

Antigen primed macrophage and thymic RNAs hybridize in an antigen restricted fashion. Thymic RNA complementary to the antigen-stimulated macrophage RNA can be obtained from mice inoculated with antigen three days earlier. The ratio of thymic and macrophage RNAs for the optimal hybridization was 50: 1. The optimal incubation time for hybridization was 32 hours at 60 d`C. Hybrid RNAs were insensitive to various kinds of RNase. The 31P Fourier transform nuclear magnetic resonance spectra identified hybrid RNA molecules from single stranded RNA's.     

Development of a DNA probe for Streptococcus bovis by using a cloned amylase gene.

Streptococcus bovis is a normal inhabitant of the rumen but has been implicated as a causative agent for ruminal lactic acidosis and related problems. While rarely isolated from humans, S. bovis has been identified as a causative agent for endocarditis, meningitis, and septicemia. Recent reports have also suggested a correlation between human colonic carcinoma and increased levels of S. bovis. Identification of S. bovis strains of human origin has been problematic because of variations in results of biochemical tests compared with results for ruminal strains. We have tested a cloned amylase gene from the ruminal strain S. bovis JB1 as a potential DNA probe for rapid and accurate identification of S. bovis strains from all sources. DNAs from strains identified as S. bovis, of both human and ruminal origin, were found to hybridize with the probe under stringent conditions. The probe also hybridized with variants of S. bovis that did not grow on starch. The probe did not hybridize with DNA isolated from other bacteria of human colonic and ruminal origin, including Bacteroides thetaiotaomicron, Bacteroides ruminicola, Butyrivibrio fibrisolvens, and Enterococcus faecalis but did demonstrate hybridization with Streptococcus salivarius.     

布氏菌544A基因片段的PCR扩增及其序列分析

利用ＰＣＲ和ＤＮＡ重组技术，成功的扩增了牛布氏菌５４４Ａ基因。回收含Ｂｒ．ａｂｏｒｔｕｓ５４４ＡＤＮＡ片段，插入ＳｍａＩ单酶切的ｐＵＣ９载体中，进行核苷酸序列分析，证实克隆的ＤＮＡ片段长度为２２４ｂｐ，在该序列中含有单个开放新闻记者框架。将重组用Ｋｐｎ１和ＢａｍＨ１双酶切下Ｂｒ．ａｂｏｒｔｕｓ５４４ａＤＮＡ片段标记探针，均与６种布氏菌杂交出现阳性结果。说明Ｂｒ．ａｂｏｒｔｕｓ５４４Ａ２２４ｂｐＤＮ     

Antigen restricted hybridization between antigen primed macrophage and thymic RNA

Antigen primed macrophage and thymic RNAs hybridize in an antigen restricted fashion. Thymic RNA complementary to the antigen-stimulated macrophage RNA can be obtained from mice inoculated with antigen three days earlier. The ratio of thymic and macrophage RNAs for the optimal hybridization was 50 : 1. The optimal incubation time for hybridization was 32 hours at 60 degrees C. Hybrid RNAs were insensitive to various kinds of RNase. The 31p Fourier transform nuclear magnetic resonance spectra identified hybrid RNA molecules form single stranded RNS's.     

Characterization of bovine septicemic, bovine diarrheal, and human enteroinvasive Escherichia coli that hybridize with K88 and F41 accessory gene probes but do not express these adhesins

Certain DNA probes derived from accessory genes of cloned K88 and F41 determinants hybridize with Escherichia coli strains that express K88 or F41 and with certain other E. coli strains that do not express these antigens. We found that these probes hybridized with human enteroinvasive E. coli, and with bovine E. coli isolates which produced a fatal septicemia in experimentally infected piglets. These strains did not hybridize with probes derived from the structural subunit genes encoding the K88 and F41 antigens. E. coli strains isolated from turkeys with septicemia, Shigella and Salmonella strains did not hybridize to the K88 and F41 accessory gene probes. The K88 and F41 accessory genes probes hybridized with a 200 kb plasmid which is required for invasion by human enteroinvasive E. coli. The K88 and F41 accessory gene homology in the bovine isolates was located on a 150 kb transmissible plasmid but was unrelated to plasmids encoding aerobactin, Vir, or colicin V, which are suspected virulence factors in septicemic E. coli. A common plasmid-encoded antigen was associated with bovine isolates that hybridized with the K88 and F41 accessory gene probes. This included strains which express CS31A, a surface antigen associated with bovine septicemic E. coli, which also hybridized with the K88 and F4 accessory gene probes. The results suggest that the K88 and F41 accessory gene probes hybridized with sequences that may be associated with a common mechanism of pilus expression in distinct groups of E. coli pathogens.     

Molecular genotyping of isolates of Actinobacillus actinomycetemcomitans by pulsed-field gel electrophoresis after separate digestion with Sse8387I and XhoI

Genomic DNA from 18 Japanese clinical isolates of Actinobacillus actinomycetemcomitans was obtained from six periodontitis patients and analyzed using pulsed-field gel electrophoresis (PFGE) after separate digestion with Sse8387I and with XhoI. Three isolates from an identical patient were found to share an identical PFGE profile, and isolates from distinct patients were found to have PFGE profiles distinctly different from each other. Consequently, the 18 Japanese clinical isolates were discriminated into six distinct genotypes by means of PFGE.The genomic DNA from the other six reference strains (ATCC33384, ATCC43717, ATCC 43718, JCM2434, JCM2435 and Nig-1) was discriminated into six genotypes by the same PFGE methodology, and these six genotypes were found to be distinctly different from the six genotypes of the 18 Japanese clinical isolates described above.Serotyping demonstrated three PFGE genotypes in the serotype a strains, four the serotype b strains and three the serotype c strains.The present results clearly suggest that the PFGE procedure after separate digestion with Sse8387I and with XhoI has an excellent discriminatory power amongst strains and has a good genotypability for A. actinomycetemcomitans.     

Expression of antigens from chromosomal and linear plasmid DNA of Borrelia coriaceae.

Three recombinant plasmids containing DNA from Borrelia coriaceae, the putative agent of epizootic bovine abortion, expressed antigens in Escherichia coli that reacted with antibodies specific for B. coriaceae. Two of the recombinants each expressed a single high-molecular-weight antigen. The third recombinant expressed three smaller antigens. The DNA inserts were sized and mapped. Hybridization of the cloned inserts to pulsed-field electrophoresis samples of B. coriaceae whole-cell DNA revealed the origin of two of the inserts to be located in linear plasmids. One of these, expressing three antigens, was located in a 210-kilobase linear plasmid. A second recombinant expressed a single antigen but hybridized to at least three distinct linear plasmids. The third clone also expressed a single antigen but was demonstrated to be chromosomal in origin.